The opioid system modulates several physiological processes including analgesia, stress response, immune response, and neuroendocrine function (Herz, Opioids Vol. 1, Springer-Verlag, Berlin, 1993). Pharmacological and molecular cloning studies have identified three opioid receptor types, delta (δ), kappa (κ), and mu (μ), that mediate these diverse effects (Miotto et al., The Pharmacology of Opioid Peptides, L. Tseng ed., 57-71, Harwood Acad. Publishers, 1995; Kieffer et al., Cell Mol. Neurobiol., 15:615-35, 1995). The opioid receptors are known to couple with pertussis toxin sensitive G-proteins.
Little is known, however, about the ability of these receptors to interact to form new functional structures, the simplest of which would be a dimer. Structural and biochemical studies reveal that other G-protein coupled receptors (GPCRs) interact to form homodimers (Herbert and Bouvier, Biochem Cell Biol., 76:1-11, 1998; Gouldson et al., Protein Eng., 11:1181-93, 1998). Moreover, non-functional GABA receptors heterodimerize to form a functional receptor, suggesting that dimerization is crucial for this receptor function (Jones et al., Nature, 396:674-679, 1998; Kaupmann, et al., Nature, 396:683-687, 1998; White et al., Nature, 396:679-682, 1998; and Kuner et al., Science, 283:7477, 1999).
It is now clear from work carried out in many laboratories over the last twenty years that there are three well-defined or “classical” types of opioid receptors: mu (μ), delta (δ), and kappa (κ). Genes encoding these receptors have been cloned (Evans et al., Science, 258:1952, 1992; Kieffer et al., Proc. Natl. Acad. Sci. USE, 89:12048, 1992; Chen et al., Mol. Pharmacol., 44:8, 1993; Minami et al., FEBS Lett., 329:291, 1993). More recently, cDNA was identified encoding an “orphan” receptor that has a high degree of homology to the “classical” opioid receptors; on the basis of its structural homology, this receptor has been classified as an opioid receptor and has been named ORL (opioid receptor-like) (Mollereau et al., FEBS Lett., 341:33, 1994). As would be predicted from their known abilities to couple through pertussis toxin-sensitive G-proteins, all of the cloned opioid receptors possess the same general structure of an extracellular N-terminal region, seven transmembrane domains and intracellular C-terminal tail structure. There is pharmacological evidence that subtypes of each receptor exist. Other types of novel, less well-characterized opioid receptors (termed ε, γ, ι, ζ) have also been postulated. The σ-receptor, however, is no longer regarded as an opioid receptor.
Opioid receptors are reviewed extensively in a publication entitled “Opioid” edited by A. Herz and in a publication from Tocris Cookson Inc. (USA)/Tocris Cookson Ltd. (UK) entitled “Opioid Receptors”, co-authored by A. Corbett, S. McKnight and G. Henderson, 1999.
μ-Receptor Subtypes
The MOR-1 gene, encoding for one form of the μ-receptor, shows approximately 50-70% homology to the genes encoding for the δ-(DOR-1), κ-(KOR-1) and orphan (ORL1) receptors. Two splice variants of the MOR-1 gene have been cloned, differing only in the presence or absence of 8 amino acids in the C-terminal tail. The splice variants exhibit differences in their rate of onset and recovery from agonist-induced internalization but their pharmacology does not appear to differ in ligand binding assays (Koch et al., N.S. Archives of Pharmacology, 357:SS44, 1998). Furthermore, in the MOR-1 knockout mouse, morphine does not induce antinociception, demonstrating that at least in this species morphine-induced analgesia is not mediated through δ- or κ-receptors (Matthes et al., Nature, 383:818, 1996). Similarly morphine does not exhibit positive reinforcing properties or an ability to induce physical dependence in the absence of the MOR-1 gene. The μ1/μ2 subdivision was proposed by Pasternak and colleagues to explain their observations, made in radioligand binding studies, that 3H-labelled-μ, -δ, and -κ ligands displayed biphasic binding characteristics (Wolozin and Pasternak, Proc. Natl. Acad. Sci. USA, 78:6181, 1981).
Several related observations suggest the existence of a yet unidentified form of μ-receptor of which analogues of morphine with substitutions at the 6 position (e.g., morphine-6β-glucuronide, heroin and 6-acetyl morphine) are agonists, but with which unsubstituted morphine itself does not interact (Rossi et al., Neuroscience Letters, 216:1, 1996).
δ-Receptor Subtypes
The DOR-1 gene is the only δ-receptor gene cloned to date. However, two, overlapping subdivisions of δ-receptor have been proposed (δ1/δ2 and δcx/δncx) on the basis of in vivo and in vitro pharmacological experiments. The subdivision of the δ-receptor into δ1 and δ2 subtypes was proposed primarily on the basis of in vivo pharmacological studies.
The δcx and δncx subdivision of δ-receptors was based on the hypothesis that one type of δ-receptor (δcx) was complexed with μ-receptors (and perhaps also κ-receptors) whereas no association with an opioid receptor complex has been observed for the other type of δ-receptor (δncx) (Rothman et al., Handbook Exp. Pharmacol., A. Herz ed., 104/1:217, 1993).
Data obtained from subsequent radioligand binding studies have been interpreted as demonstrating the existence of further subtypes of the δncx receptor, i.e., δ(ncx−1) and δ(ncx−2). More recently, it has been suggested that the δ(ncx−1) receptor is in fact identical to the δ1-receptor and the δcx-receptor is identical to the δ2-receptor of the previous classification (Xu et al., Peptides, 14:893, 1993).
κ-Receptor Subtypes
The situation regarding the proposals for subtypes of the κ-receptor is rather more complex than for the μ- and δ-receptors, perhaps because of the continuing use of non-selective ligands to define the putative sites. The evidence for the need for sub-division of the κ-receptor comes almost entirely from radioligand binding assays.
Studies of 3H-ethylketocyclazocine 3H-EKC binding in guinea-pig spinal cord pointed to the existence of a non-homogeneous population of high-affinity binding sites, and led to the first proposal for κ1- and κ2-sites distinguished by their sensitivity to DADLE (Attali et al., Neuropeptides, 3:53, 1982).
Subdivision of the κ1-site in guinea-pig brain into κ1a and κ1b, was proposed to resolve the complex displacement of either 3H-EKC or 3H-U-69,593, with dynorphin B and α-neo-endorphin which both preferentially bound to the proposed κ1b sub-subtype (Clark et al., J. Pharmacol. Exp. Ther., 251:461, 1989). The same study proposed the existence of κ3 subtype, insensitive to U-50,488, that was identified from the binding of 3H-naloxone benzoylhydrazone. The pharmacology of this later “κ3-site” is rather different from the κ3/MRF site of bovine adrenal medulla, and has been proposed to be the receptor mediating the antinociceptive effect of nalorphine, termed Martin's “N”-receptor (Paul et al., J. Pharmacol. Exp. Ther., 357:1, 1991).
Definitive functional pharmacological evidence supporting the existence of this confusing number of putative subtypes of the κ-receptor is lacking, because of the absence of subtype-specific antagonists.
All of this uncertainty and confusion about the precise identity of opioid receptors, and the number of different receptors, has hampered efforts to identify more effective, more specific opioid agonists and antagonists, i.e., more specific drugs with fewer untoward side effects, within a large family of neuropharmaceuticals including narcotic analgesics.
Thus, there is a need in the art to identify the molecular basis for the diversity of opioid receptor specificities.
There is a further need to identify specific opioid receptors for screening and development of more effective, less addictive, narcotics.
The present invention addresses these and other needs in the art.